Varicella-zoster virus (VZV) is a medically important human -herpesvirus that causes varicella (chickenpox) and leads to zoster (shingles) upon reactivation from latently infected sensory ganglia. Varicella can be serious and is life-threatening in immunocompromised patients. VZV exhibits tropism for T cells, skin and neurons during infection of the human host and overcomes the usual constraint against fusion between fully differentiated host cells to form multinucleated polykaryocytes, a hallmark of VZV pathogenesis. Glycoprotein B (gB) along with the gH/gL heterodimer is known to be critical for fusion of the virion envelope with the target cell membrane during herpesvirus entry. Our novel concept is that VZV mediates cell-cell fusion through a gB-dependent intracellular signaling function. This is based on our new evidence that preventing tyrosine phosphorylation of the gB cytoplasmic domain (gBcyt) leads to anomalies in cell-cell fusion and syncytia formation in vitro. Our application will investigate how the gBcyt modulates cell- cell fusion mechanisms via intracellular signaling pathways to produce the characteristic syncytia in vitro and fusion of epidermal cells and neuron-satellite cells caused by VZV infection of skin and ganglia in vivo. In Aim 1 we will determine how VZV modifies cellular regulation to favor transcription of genes that facilitate cell-cell fusion and syncytia formation by applying the high-throughput whole-transcriptome sequencing technology, RNA-seq, to our new fusion assay and our virus mutants, which carry mutations in the gBcyt residues that affect cell-cell fusion. To quantify the effects o tyrosine phosphorylation, the spatiotemporal evolution of syncytia formation will be measured in real-time for VZV and the gBcyt mutants. To determine the role of genes in cell fusion, as identified by RNA-seq, we will perform gene perturbation experiments to assess their biological significance in the context of VZV replication. Aim 2 will determine how the gBcyt regulates intracellular signaling events in cell fusion via post-translational modifications of tyrosine and/r lysine residues by cellular or viral proteins. Mass spectrometry will be used to identify cellular and viral proteins that interact with the gBcyt domain in its tyrosine-phosphorylated and non-phosphorylated forms. Lysine mutagenesis studies will be performed to assess the effects of acetylation and ubiquitination posttranslational modifications on VZV fusion and virulence. Finally, Aim 3 will establish whether the gBcyt modulates polykaryocyte formation to optimize VZV infection of skin and DRG. Our mutant viruses will be compared to wild type VZV for replication competencies in human skin and neuronal tissue using novel reporter viruses. We will establish the role of newly identified genes required for cell-cell fusion using a novel shRNA carrying virus. Given the significance of polykaryocyte formation for pathogenesis, deciphering how VZV regulates this process has the potential to yield new strategies for vaccine virus attenuation and antiviral drug design to ease the burden on vulnerable populations.